This invention relates to a microfabricated capillary electrophoresis chip for detecting multiple redox-active labels simultaneously using a matrix coding scheme and to a method of selectively labeling analytes for simultaneous electrochemical detection of multiple label-analyte conjugates after electrophoretic or chromatographic separation.
Capillary Electrophoresis (CE) is proving to be a powerful tool for DNA-sequencing and fragment sizing due to its low sample volume requirements, higher efficiency and rapidity of separations compared to the traditional approach of slab gel electrophoresis (Swerdlow, H. and Gesteland, R., (1990) Nucl. Acid. Res. 18, 1415-1419) (Kheterpal, I., Scherer, J. R., Clark, S. M., Radhakrishnan, A., Ju. J., Ginther, C. L., Sensabaugh, G. F. and Mathies, R. A., (1996) Electrophoresis 17, 1852-1859). More recently, microfabricated CE devices and Capillary Array Electrophoresis (CAE) microplates have demonstrated their potential for rapid, parallel separation of DNA sizing and sequencing samples (Woolley, A. T. and Mathies, R. A., (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11348-11352) (Woolley, A. T. and Mathies, R. A., Anal. Chem. 67, 3676-3680, 1995) (Woolley, A. T., Sensabaugh, G. F., and Mathies, R. A., (1997) Anal. Chem. 69, 2256-2261) (Simpson, P. C., Roach, D., Woolley, A. T., Thorsen, T., Johnston, R., Sensabaugh, G. F. and Mathies, R. A., (1998) Proc. Natl. Acad Sci. U.S.A. 95, 2256-2261). The development of these miniaturized CE platforms has been driven by the concept of making fully integrated, inexpensive and portable analytical systems.
Electrochemical (EC) detection is an approach which is easily adaptable to miniaturized CE platforms without any sacrifice in sensitivity or selectivity. EC detection has been widely used with conventional CE in fused-silica capillaries for highly sensitive and selective detection of various analytes. A critical problem in this application is figuring out how to decouple the high electrophoretic separation currents from the electrochemical detection system. Wallingford and Ewing first described the use of an on-column fracture with a porous glass-flit to decouple the high electrophoresis currents from the small electrochemical signals (Wallingford, R. A. and Ewing, A. G., (1987) Anal. Chem. 59, 1762-1766). The porous frit provided a way to ground the electrophoresis current prior to the detector electrode which was poised at the outlet of the capillary. The analytes in the buffer were pumped to the detector electrode by the residual electroosmotic flow existing in the capillary. Due to effective decoupling of the separation electric field from the detector electrode, this scheme allowed highly sensitive detection of the analytes. However, this system was very fragile due to the delicate porous glass frit, and it was difficult to align the electrode at the outlet of the capillary. A number of other designs have since been used to isolate the electrophoresis current which include porous nafion tubing (O""Shea, T. J., Greenhagen, R. D., Lunte, S. M., Lunte, C. E., Smyth, M. R., Radzik, D. M. and Watanabe, N., (1992) J. Chromatogr. 593, 305-312), and palladium joints (Kok, W. T., and Sahin, Y., (1993) Anal. Chem. 65, 2497-2501). All these designs are very fragile and not amenable for the construction of a robust CE-EC system. End-column detection in small diameter capillaries was then proposed as an alternative to the on-column fracture designs (Huang, X. H., Zare, R. N., Sloss, S., and Ewing, A. G., (1991) Anal. Chem. 63, 189-192). This approach capitalized on the fact that smaller inner diameter ( less than 10 xcexcm) capillaries exhibit very low electrophoretic currents due to their much smaller area. Thus, no isolation was required for the electrophoresis current, thereby obviating the need for any on-column current decouplers. EC detection has been successfully used as a detection method for capillary electrophoresis in fused-silica capillaries as small as 2 xcexcm in diameter (Olefirowicz, T. M. and Ewing, A. G., (1990) Anal. Chem. 62, 1872-1876), with detection limits for various analytes in the femtomole to attomole mass range. Smaller diameter electrophoretic capillaries require the use of smaller diameter electrodes, or microelectrodes. Background noise is lower at these microelectrodes due to a sharp decrease in background charging currents (Bard, A. J. and Faulkner, L. R., (1980) Electrochemical Methods:Fundamentals and Applications, New York, John Wiley and Sons). This leads to better concentration sensitivity due to the higher signal-to-noise ratio. Mass sensitivity is also enhanced at these microelectrodes over bigger electrodes due to higher coulometric efficiency (Huang, X. H. et al., supra). End-column detection therefore allows the CE-EC approach to be performed successfully without any loss in sensitivity. However, very expensive micropositioners are required in order to accurately position microelectrodes at the outlet of such small diameter capillaries. Consequently, run-to-run reproducibility is very poor using this design. Many researchers have tried various ways of gluing an electrode in place outside a CE-capillary (Fermier, A. M., Gostkowski, M. L., and Colon, L. A., (1996) Anal. Chem. 68, 1661-1664) (Chen, M. C. and Huang, H. J., (1997) Anal. Chem. Acta. 341, 83-90), but this approach is still very tedious and irreproducible with very small diameter capillaries. It is also very hard to reliably make a large number of such assemblies with the same capillary-electrode alignment. Thus, end-column detection with conventional capillaries and electrodes is not useful for routine and automated analyses.
Microfabrication of electrodes at the outlet of microfabricated channels makes it possible to routinely perform end-column detection as the electrodes can be permanently fabricated with high precision and reproducibility. An approach to CE-EC detection on fully microfabricated systems was illustrated recently by Woolley et al. (Woolley, A. T., Lao, K., Glazer, A. N. and Mathies, R. A., (1998) Anal. Chem. 70, 684-688). Platinum microelectrodes were fabricated at the outlet of a CE-channel etched in a glass plate so as to allow effective isolation of the detection system from the electrophoresis currents in an end-column detection format. Sensitive detection of neurotransmitters with detection limits in the attomole range was accomplished with high reproducibility. The feasibility of using microfabricated capillary electrophoresis chips with integrated electrochemical detection to perform high-sensitivity DNA restriction fragment analysis and PCR product sizing was also demonstrated. DNA fragments were indirectly detected by adding an electroactive intercalator, iron-phenanthroline in the electrophoresis buffer. A xcfx86x HAE-III restriction digest was detected using this approach. The detection limit for the 603 base pair (bp) fragment was around 30 zeptomoles, and a PCR product from Salmonella was sized easily against an internal restriction fragment standard. This illustrates that microfabricated CE-EC systems are capable of highly sensitive detection.
However, indirect detection is not suitable for the selective detection of certain typical analytes in a complex mixture, as it is not specific to the detection of the desired analytes. Furthermore, since there was only one indirect and non-covalent redox active label in our previous work, it was more difficult to compare the size of an unknown DNA fragment with that or a standard because the signals can overlap. Direct labeling of analytes is typically needed to achieve selective simultaneous multiplex detection of various analytes. For example, in the case of fluorescence based DNA-sequencing, four different fluorescent labels were used required for the simultaneous detection of each of the four base termination ladders generated using the Sanger dideoxy method (Sanger, F., Nicklen, S., and Coulson, A. R., (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467) (Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S. B. and Hood, L. E., (1986) Nature 321, 674-679) (Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A. and Baumeister, K., (1987) Science 238, 336-341). In these studies, unique fluorescent labels were linked to the four different sequencing fragment ladders by either labeling the primer or the terminator used in the extension reaction with unique labels. Furthermore, U.S. Pat. No. 5,436,130 describes a DNA sequencing method which uses single slab gel lane or electrophoresis capillary. Sequencing fragments are separated in said lane and detected using a laserexcited, confocal fluorescence scanner. In this case, each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels to uniquely label the four sets of sequencing fragments. Also described is a method of using radio-isotope labels to similarly code or label the fragments. For DNA sequencing applications, it would clearly be valuable to develop methods for multiplex electrochemical labeling, separation and detection. It would also be valuable to develop analogous methods for the multiple labeling of DNA fragments to be used in DNA diagnostics employing RFLP, STR or SNP assays and the like. The simultaneous detection of multiple labels in a CE-EC run requires the development of strategies which are capable of detecting the multiple electrochemical signals generated by such a system. Differences in redox potentials between different compounds can be exploited for selective measurements using EC detection. Traditional voltammetric methods have been widely used in the literature to exploit these differences (Kristensen, E. W., Kuhr, W. G., and Wightman, R. M., (1987) Anal. Chem. 59, p. 1752). But, these methods involve rapid scanning of the electrode potential, which leads to large background charging currents. Poor sensitivity is obtained due to high background noise caused by these large charging currents.
It is a general object of the present invention to provide a method and apparatus for simultaneous detection of multiple electrochemical signals using multiple electrodes.
It is another object of the present invention to provide a microfabricated CE chip having multiple electrodes with each electrode optimized for the detection of a specific label or analyte.
It is a further object of the present invention to provide a method and apparatus for multiplex labeling and electrochemical detection of multiple analytes.
It is a still further object of the present invention to provide a method for attaching redox labels to analytes, electrophoretically separating the analytes and electrochemically detecting the individual separated analytes.
Another object is achieved by developing methods to label multiple analytes with different EC-labels that can be distinctly detected.
The foregoing and other objects of the invention are achieved by a microfabricated electrophoresis chip which includes a separation channel which widens into a detection reservoir with a plurality of thin film detecting or working electrodes extending into said detection reservoir closely adjacent the end of the separation channel. Another object of the invention is achieved by a method of simultaneously detecting electrochemical signals generated at said detection electrodes by different redox labels attached to analytes in a mixture of analytes after the analytes have been separated in the electrophoresis chip.
Another object is the method of multiplex electrochemical labeling and coding of various analytes to simultaneously distinguish multiple analytes simultaneously.
An additional object is to develop separation, detection and labeling methods for performing genotyping and sequencing with electrochemical detection.